Influence of reduced glutathione on end-joining of DNA double-strand breaks: Cytogenetical and molecular approach.

Radiation induced DNA double-strand breaks (DSB) are the major initial lesions whose misrejoining may lead to exchange aberrations. However, the role of glutathione (GSH), a major cellular thiol, in regulating cell's sensitivity to DNA damaging agents is not well understood. Influence of endogenous GSH on the efficiency of X-rays and bleomycin (Blem) induced DNA DSBs end-joining has been tested here cytogenetically, in human lymphocytes and Hct116 cells. In another approach, oligomeric DNA (75bp) containing 5'-compatible and non-compatible overhangs mimicking the endogenous DSB were for rejoining in presence of cell-free extracts from cells having different endogenous GSH levels. Frequency of aberrations, particularly exchange aberrations, was significantly increased when Blem was combined with radiation. The exchange aberration frequency was further enhanced when combined treatment was given at 4°C since DNA lesions are poorly repaired at 4°C so that a higher number of DNA breaks persist and interact when shifted from 4°C to 37°C. The exchange aberrations increased further when the combined treatment was given to Glutathione-ester (GE) pre-treated cells, indicating more frequent rejoining of DNA lesions in presence of higher cellular GSH. This is further supported by the drastic reduction in frequency of exchange aberrations but significant increase in incidences of deletions when combined treatment was given to GSH-depleted cells. End-joining efficiency of DNA DSBs with compatible ends was better than for non-compatible ends. End-joining efficiency of testicular and MCF7 cell extracts was better than that of lungs and Hct116 cells. Cell extract made from GE-treated MCF-7 cells provided more efficient end-joining than from untreated and GSH-depleted cells. However, direct addition of GSH to the cell-free extracts showed considerable reduction in end-joining efficiency. Present data indicate that higher endogenous GSH favours rejoining of DNA DSBs (both restitution and illegitimate reunion) which in turn produce more exchange aberrations.

Interaction of DNA-lesions induced by sodium fluoride and radiation and its influence in apoptotic induction in cancer cell lines.

Fluoride is an essential trace element but also an environmental contaminant with major sources of exposure being drinking water, food and pesticides. Previous studies showed that sodium fluoride (NaF) at 5 mM or more is required to induce apoptosis and chromosome aberrations and proposed that DNA damage and apoptosis play an important role in toxicity of excessive fluoride. The aim of this study is directed to understand the nature of DNA-lesions induced by NaF by allowing its interaction with radiation induced DNA-lesions. NaF 5 mM was used after observing inability to induce DNA damages and apoptosis by single exposure with 50 µM or 1 mM NaF. Co-exposure to NaF and radiation significantly increased the frequency of aberrant metaphases and exchange aberrations in human lymphocytes and arrested the cells in G1 stage instead of apoptotic death. Flow cytometric analysis, DNA fragmentation and PARP-cleavage analysis clearly indicated that 5 mM NaF together with radiation (1 Gy) induced apoptosis in both U87 and K562 cells due to down regulation of expression of anti-apoptotic proteins, like Bcl2 in U87 and inhibitors of apoptotic proteins like survivin and cIAP in K562 cells. This study herein suggested that single exposure with extremely low concentration of NaF unable to induce DNA lesions whereas higher concentration induced DNA lesions interact with the radiation-induced DNA lesions. Both are probably repaired rapidly thus showed increased interactive effect. Coexposure to NaF and radiation induces more apoptosis in cancer cell lines which could be due to increased exchange aberrations through lesions interaction and downregulating anti-apoptotic genes.

Induction of chromosome instability and stomach cancer by altering the expression pattern of mitotic checkpoint genes in mice exposed to areca-nut.

BACKGROUND: There are strong indications for a causal association between areca-nut consumption and cancers. In Meghalaya, India, the variety of areca-nut is used as raw and unprocessed form whose chemical composition and pharmacological actions have been reported. Yet we know little on the initial pathway involved in areca-nut associated carcinogenesis since it is difficult to assess its effects on genetic alterations without interference of other compounding factors. Therefore, present study was undertaken in mice to verify the ability of raw areca-nut (RAN) to induce cancer and to monitor the expression of certain genes involved in carcinogenesis. This study was not intended to isolate any active ingredients from the RAN and to look its action. METHODS: Three groups of mice (n = 25 in each) were taken and used at different time-points for different experimental analysis. The other three groups of mice (n = 15 in each) were considered for tumor induction studies. In each set, two groups were administered RAN-extract ad libitum in drinking water with or without lime. The expression of certain genes was assessed by conventional RT-PCR and immunoblotting. The mice were given the whole RAN-extract with and without lime in order to mimic the human consumption style of RAN. RESULTS: Histological preparation of stomach tissue revealed that RAN induced stomach cancer. A gradual increase in the frequency of precocious anaphase and aneuploid cells was observed in the bone marrow cells with a greater increment following RAN + lime administeration. Levels of p53, Bax, Securin and p65 in esophageal and stomach cells were elevated during early days of RAN exposure while those of different mitotic checkpoint proteins were downregulated. Apoptotic cell death was detected in non-cancerous stomach cells but not in tumor cells which showed overexpression of Bax and absence of PARP. CONCLUSION: Present study suggested (a) RAN induces stomach cancer, however, presence of lime promoted higher cell transformation and thereby developed cancer earlier, (b) perturbations in components of the chromosome segregation machinery could be involved in the initial process of carcinogenicity and (c) the importance of precocious anaphase as a screening marker for identification of mitotic checkpoint defects during early days.

Comparison of effectiveness in antitumor activity between flavonoids and polyphenols of the methanolic extract of roots of Potentilla fulgens in breast cancer cells.

Potentilla fulgens has been used for a long time as folk remedy for a variety of ailments without having information on its pharmacological action. The study was intended to determine the effectiveness in antitumor activity among kaempferol (KMP) as flavonoids, ellagic acid (ELA) as polyphenols and methanolic extract of the root of P. fulgens (PRE) in Ehrlich ascites tumour (EA) and MCF-7 cancer cells. The total phenolic and flavonoid content were found to be 138.8 ± 1.6 mg gallic acid and 401.6 ± 4.6 mg quercetin per 1 gm of the extract, respectively. The extract resulted in increasing in vivo survivality of mice bearing EA cells and loss of cell viability in a dose-dependent manner in MCF-7 cells. This effect may be attributed to apoptotic cell death as confirmed by flow cytometric analysis and PARP1 proteolysis. Such induction of apoptosis was achieved by suppression of inhibitor of the apoptosis protein survivin. It was observed that endogenous level of glutathione was depleted significantly in MCF-7 cells after the treatment with PRE only but not with KMP or ELA. This induction of apoptotic cell death and lowering the level of glutathione are highly desirable mode for an anticancer agent.